Heart failure represents a major cause of morbidity and mortality among veterans. Cardiac myocyte loss due to apoptosis plays a significant role in the progression of heart failure. A major goal of my laboratory is to investigate the role of osteopontin (OPN), a matricellular protein, in the development of heart failure. Normal heart expresses low levels of OPN. OPN expression increases markedly under a variety of pathophysiological conditions of the heart. Increased OPN expression associates with increased myocyte apoptosis and myocardial dysfunction. Using streptozotocin-induced model of diabetic cardiomyopathy, we have provided evidence that increased OPN expression in myocytes associates with myocyte apoptosis and myocardial dysfunction. The molecular signals involved in OPN-stimulated cardiac myocyte apoptosis have not yet been investigated. Our preliminary data show that OPN induces apoptosis in adult rat ventricular myocytes. Neutralization of CD44 (one of the receptors for OPN) inhibited OPN-stimulated apoptosis. OPN activated merlin (a linker protein between transmembrane proteins and the actin-cytoskeleton), Mst1 (mammalian sterile 20-like kinase 1), JNKs, and increased levels of cytosolic cytochrome c. Adenoviral-mediated expression of constitutively active merlin activated Mst1, JNKs and induced myocyte apoptosis. Inhibition of JNKs or DRP1 (dynamin related protein 1; a protein involved in mitochondrial fission) inhibited OPN-stimulated apoptosis. OPN treatment increased expression of Gadd-153 and activated caspase-12, suggesting a role for OPN in induction of endoplasmic reticulum (ER) stress. RT2-qPCR Array analysis identified increased expression of Bik, a p53-regulated BH3-only pro-apoptotic member of Bcl-2 family capable of communicating between ER and mitochondria, in OPN-treated myocytes. Inhibition of JNKs or p53 inhibited OPN-stimulated increases in Bik expression. In vivo, expression of OPN in the adult mouse heart in a cardiac myocyte-specific manner increased myocyte apoptosis and myocardial dysfunction. We hypothesize that OPN acting via CD44 receptor activates Merlin, and activation of merlin induces ER stress and mitochondrial death pathway via the involvement of Mst1/JNK/p53/Bik pathway. Aim 1 will determine receptor/s for OPN in myocytes, and test the hypothesis that OPN acting via CD44 receptor induces ER stress, mitochondrial death pathway, and apoptosis. Aim 2 will test the hypotheses that interaction of OPN with CD44 receptor activates Merlin, and activation of merlin plays a pro-apoptotic role. Aim 3 will test the hypothesis that activation of merlin plays a pro-apoptotic role via the involvement of Mst1/JNKs/p53/Bik pathway. Aim 4 will address, in vivo, the role of OPN and CD44 in myocyte apoptosis and myocardial dysfunction using inducible transgenic mouse system that permits myocyte-specific expression of OPN in the heart, and using OPN and CD44 knockout mice. The proposed studies are vital in defining the role of OPN in myocyte apoptosis and myocardial dysfunction, and may uncover novel strategies for the treatment of heart failure.